Tech Notes

Q. How do I filter on Fold-Change with my two-color data?

Contrarily to a one-color experiment, two-color data can integrate both control and treated samples on the same slide. Therefore, a two-color experiment is imported as a single sample, containing both control and treated. The "Filter on Fold Change" option is thus not an appropriate choice in that case.

After data import, two-color data are typically normalized by taking the ratio between one channel and the other. The normalized value is then Cy5/Cy3, or equivalent, and represents the fold-change between the two channels. If you wish to filter on fold-change between Cy5 and Cy3, choose "Filter on Expression Level" from the Filtering menu. Select "Normalized" value to filter upon, as the normalized value is the ratio between the two channels.

Examples

1. Cy3 channel is the "Control" and the Cy5 channel is the "Treatment" sample in your experiment. You wish to filter on genes with a fold change of 2 in the treatment, compared to the control channel, in other words, those that are two-fold over expressed due to treatment. Select "Filter on Expression Level" from the Filtering menu, and select to filter on "normalized" values. Enter "2" as minimum value to filter on, the minimum number of conditions this value should appear in, and click "Start".
   
   
2. You wish to filter on genes that are 2 fold downregulated in the treatment, compared to the control (see example 1). Enter "0.5" as maximum value to filter on, the minimum number of conditions this value should appear in, and click "Start".
   
   
   
   
3. Cy3 is a common reference, and you wish to filter on fold change between two conditions on two different slides. In that case, choose "Filter on Fold Change" from the Filtering menu. Select condition 1 and 2 from the desired interpretation, and select the appropriate fold-change.
   
   
   
   

In this example, both Heatshock and Radiation treatment samples are hybridized with Cy5. This filter will allow you to find genes with a 2-fold differential expression between heatshock and radiation treatment.